U.S. patent application number 10/760000 was filed with the patent office on 2004-07-29 for unitary device with internal microscopic counting grid used for analysis of microscopic particles contained in liquid.
Invention is credited to Qiu, Jean.
Application Number | 20040145805 10/760000 |
Document ID | / |
Family ID | 32738323 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040145805 |
Kind Code |
A1 |
Qiu, Jean |
July 29, 2004 |
Unitary device with internal microscopic counting grid used for
analysis of microscopic particles contained in liquid
Abstract
A unitary counting device for cells and other microscopic
particles is described in this invention. The invention also
relates to the method of producing the counting device and to the
method of using the device. The counting device is constructed with
a top part, a connection layer, a base part, and a grid of
microscopic lines that are built inside a counting chamber to
define counting areas. Furthermore, the counting chamber has a
sample introduction port and an air escape port. A connection layer
bonds the top part and the base along perimeter of the chamber and
maintains gap uniformity. The connection layer consists of a
polymer film sandwiched by two layers of pressure sensitive
adhesive. Grid lines are fabricated using polymerizable solutions,
for narrow and thin lines, which enable counting small particles
under high magnifications.
Inventors: |
Qiu, Jean; (Andover,
MA) |
Correspondence
Address: |
Jean Qiu
1 Copley Drive
Andover
MA
01810
US
|
Family ID: |
32738323 |
Appl. No.: |
10/760000 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60440364 |
Jan 16, 2003 |
|
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Current U.S.
Class: |
359/398 ;
359/396 |
Current CPC
Class: |
B01L 3/502707 20130101;
G01N 15/1456 20130101; B01L 2300/0822 20130101; G02B 21/34
20130101 |
Class at
Publication: |
359/398 ;
359/396 |
International
Class: |
G02B 021/34; G01N
021/01 |
Claims
What is claimed is:
1. A unitary chamber for counting microscopic objects in liquid,
comprising a top part, a base part, a counting grid, a connecting
layer, a sample introduction port, and an air escape port, wherein
said connecting layer is between said top part and said base part,
and wherein said connecting layer is at a pre-determined
thickness.
2. The unitary chamber as defined in claim 1, wherein said counting
grid is an integral part of said top part.
3. The unitary chamber as defined in claim 2, wherein said counting
grid is on the bottom side of the said top part.
4. The unitary chamber as defined in claim 1, wherein said counting
grid is an integral part of said base part.
5. The unitary chamber as defined in claim 4, wherein said counting
grid is on the top side of the said bottom part.
6. The unitary chamber as defined in claim 1, wherein said sample
introduction port and said air escape port are integral parts of
said top part.
7. The unitary chamber as defined in claim 1, wherein said counting
grid line width range from 0.1 micrometer to 1 mm.
8. The unitary chamber as defined in claim 1, wherein said counting
grid line width range from 1 micrometer to 25 micrometer.
9. The unitary chamber as defined in claim 1, wherein said counting
grid line thickness ranges from 0.1 micrometer to 50
micrometer.
10. The unitary chamber as defined in claim 1, wherein said
counting grid is made by polymerizable solution.
11. The unitary chamber as defined in claim 1, wherein said
counting grid is made by the radiation polymerizable solution.
12. The unitary chamber as defined in claim 1, wherein said
counting grid is made by the ultraviolet light polymerizable
solution.
13. The unitary chamber as defined in claim 1, wherein said
counting grid is made by polymerizable solution onto the bottom
side of the top part.
14. The unitary chamber as defined in claim 1, wherein said
counting grid is made by polymerizable solution onto the top side
of the base part.
15. The unitary chamber as defined in claim 1, wherein said
connecting layer is made with adhesives dispersed with gap defining
particles.
16. The unitary chamber as defined in claim 1, wherein said
connecting layer consists of only pressure sensitive adhesives.
17. The unitary chamber as defined in claim 1, wherein said
connecting layer consists of a plastic film, sandwiched by two
layers of pressure sensitive adhesives.
18. A unitary device with plurality of unitary chambers as defined
in claim 1.
19. A method of making a unitary chamber for counting microscopic
objects in liquid, said chamber comprising a top part, a base part,
a counting grid, a connecting layer, a sample introduction port,
and an air escape port, wherein said connecting layer is between
said top part and said base part, and wherein said connecting layer
is at a pre-determined thickness.
20. A method of counting microscopic objects in liquid with a
unitary chamber, said chamber comprising a top part, a base part, a
counting grid, a connecting layer, a sample introduction port, and
an air escape port, wherein said connecting layer is between said
top part and said base part, and wherein said connecting layer is
at a pre-determined thickness.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application 60/440,364, titled "Disposable cell counter", filed on
Jan. 16, 2003.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally relates to devices for
counting microscopic particles in solution. In particular, the
microscopic particles may be cells or other biological objects.
[0004] 2. Description of the Prior Art
[0005] Biological solutions, such as blood, spinal fluid, cell
culture and urine, are routinely analyzed for their microscopic
particle concentrations. As an example, for determining cell
concentrations in biological solutions, a commonly used method is
to spread the cell-containing solution into a thin layer without
cell overlap in the vertical direction. A precise volume is
determined by keeping the height of the solution at a known
constant level. Cells are viewed under an optical microscope and
enumerated in defined areas. To eliminate the variation caused by
microscopes, an area-defining grid is preferred in the counting
chamber. A commonly used cell counting device is called
hemacytometer, as disclosed in U.S. Pat. No. 1,693,961 and U.S.
Pat. No. 2,039,219.
[0006] The hemacytometer is a thick glass slide with three raised
platforms. The central platform is lower than the two outer
platforms by a given height, which is typically less than 1 mm. The
central platform has precisely ruled grid lines etched into glass.
A cover slip is placed on top of the raised platforms. The cover
slip rests on the two outer and higher platforms, thus a gap is
form between the cover slip and the central platform. When liquid
is introduced into the gap between the cover slip and the central
platform, the grid that is etched into the central platform defines
sections for the particle-containing liquid. Using an optical
microscope, particles are counted inside the counting grid.
Concentration of particles can be obtained by calculation using the
liquid volume within the grid and number of particles counted
within the grid.
[0007] Before using a hemacytometer, the cell suspension is diluted
so that the cells do not overlap each other on the grid, and that
cells are uniformly distributed in the counting area. To load the
counting chamber, the first step is to place the cover slip over
the counting surface. Then the suspension is introduced into the
area under the cover slip by capillary action through the side of
the counting area. This can done with a pipette. Enough liquid must
be introduced so that the central platform surface is just covered.
(If too much liquid is introduced, liquid overflows the counting
area. When this happens, the hemacytometer must be cleaned and
dried with lens tissue, and reloaded.) The charged counting chamber
is then placed on a microscope stage and the counting grid is
brought into focus for counting.
[0008] Immediately after use, the cover slip is removed. Both the
cover slip and the raised platforms are cleaned with water or with
a mild cleaning solution, and then dried carefully.
[0009] In practice, it is desired to have a pre-assembled chamber
that eliminates the error produced when the cover slip is misplaced
or the chamber is over or under charged.
[0010] It is also desired to have a counting chamber with internal
grid to eliminate the variation in area by different microscopes.
It is further desired to have a counting chamber that is disposable
after single use to eliminate the biohazard generated during
washing hemacytometer.
[0011] Pre-assembled plastic slides with examination chambers
formed between a base plate and cover plate is taught in U.S. Pat.
No. 4,637,693. One side of the counting chamber is open to a liquid
loading area. When liquid is placed in the loading area, capillary
force draws the particle containing solution into the counting
area. The disadvantage of connecting sample loading area with the
counting area is the lack of support along the opening. During
loading, the surface tension of the liquid may draw the top of the
counting area towards the bottom, creating thickness variation,
which contributes to counting inaccuracy.
[0012] In order to hold the top part and the bottom part of the
chamber parallel, it is desired to have height defining walls
surrounding the counting chamber entirely. Such devices without
counting grid are disclosed in U.S. Pat. No. 4,441,793.
[0013] Pre-assembled plastic slides with examination chambers
formed between a base plate and cover plate with raised lines 0.012
mm to 0.023 mm width and 0.008 mm height is taught in U.S. Pat. No.
4,997,266. To count small particles with sizes less than 1 to 10
micrometers, it is necessary to use high magnifications on the
microscope, which requires much narrower lines to form the counting
grid with much smaller areas. It is also necessary to obtain grid
lines with less thickness than what is disclosed in U.S. Pat. No.
4,997,266, in order to reduce the influence of grid lines on
uniform particle distribution during the sample loading
process.
[0014] As evident from the discussion in this section, it would be
desirable and advantageous to devise a cell and particle counting
device that has a pre-assembled chamber with counting grids that
are suitable for counting small particles and with improved loading
accuracy than what is done with prior art. The pre-assembled device
eliminates the error produced when the cover slip is misplaced or
the chamber is over charged or under charged.
SUMMARY OF THE INVENTION
[0015] The present invention provides a unitary, single-use
counting chamber, for cells and other microscopic particles, with
support walls around the entire chamber and with a high-resolution
and fine-line counting grid.
[0016] In this invention, the novel disposable cell counting device
is an enclosed counting chamber with two ports for sample
application and air escape respectively. The two ports may be holes
through the flat top of the counting chamber. The flat top part is
held to a rigid and flat base part at a defined gap. Either the top
part of the chamber or the base part of the chamber contains
precisely spaced lines in a grid pattern. Both the top part and the
base part are transparent, allowing cell counting in either
transmission or reflection mode. The grid design is similar to the
well-known Improved Neubauer pattern, as commonly used in
hemacytometer. When the cell-containing solution is introduced into
the counting chamber through one of the ports with a pipette, air
escapes through the opposing port, leaving an evenly distributed
liquid layer without air bubble. The particles are counted under an
optical microscope. The particle concentration is calculated using
the volume of liquid under the counting grid. The counting device
is disposed after use.
[0017] The thickness of the liquid layer, which is confined within
the counting area with the grid, is defined by the gap between the
top and the base of the chamber. For counting accuracy, it is
essential to maintain the liquid thickness constant. The present
invention employs a supporting wall around the entire grid
area.
[0018] The supporting wall around the counting area is formed by a
connecting part, which surrounds the counting area. The purpose of
the connecting part is to provide a constant gap between the top
and the base parts of the chamber, as well as to bond the top part
and the base part together to form an enclosed chamber.
[0019] The connecting part can be formed using pressure sensitive
spacer adhesives. The spacer adhesive consists of a polymer film
sandwiched by two layers of pressure sensitive adhesives. It serves
purposes of bonding the top and the base parts of the counting
chamber together as well as providing defined chamber height.
[0020] Most living cells give very little contrast in bright field
microscopy because they have too little contrast to the solution
surrounding them and they have no color. However the various
organelles show wide variation in refractive index, which is the
tendency of the materials to bend light. The phase contrast
microscopy employs an optical mechanism to translate minute
variations in phase into corresponding changes in amplitude, which
can be visualized as differences in image contrast. With phase
contrast microscopy, living cells and microorganisms are easily
counted and characterized.
[0021] Plastic films that bear microscopic features are produced
using polymerizable solutions. It was discovered by this inventor
that the features in the film generate sufficient contrast under
phase contrast microscope, even for features less than 3 micrometer
wide and less than 0.5 micrometer thick. Using micro-replicated
plastic film to produce counting grid allows (1) the cost of
manufacturing to be low, and (2) the manufacturing of fine grid
lines, which enables measurement of small particles less than 5
micrometer in diameter. The fine grid lines remove distortion of
particle distribution during sample loading.
[0022] The present invention also describes the counting chamber
with no phase distortion by the materials themselves. This property
allows the phase contrast microscopy to be employed for particle
evaluation.
[0023] The disposable nature of devices in the present invention
allows the elimination of the cleaning procedure used with
conventional hemacytometer. No cleaning simplifies the counting
operation, as well as reduces the hazardous liquid waste, which may
be biological or radioactive.
[0024] The procedure of particle counting in this invention
consists of the following steps: (1) pipette cell-containing
solution into counting chamber, (2) count cells, and (3) dispose
used counting chamber.
[0025] The present invention is intended for counting cells and
other microscopic particles in solution, such as spores, bacteria,
and other microscopic organisms. Where appropriate, the term "cell"
is used to represent cell and any other microscopic particle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention will best be understood by reference to the
accompanying drawings, wherein:
[0027] FIG. 1 contains schematic drawings of the disposable cell
counting chamber, where FIG. 1(a) depicts the complete assembly,
FIG. 1(b) shows the interior of the counting chamber by removing
the top part, FIG. 1(c) represents top view and FIG. 1(d)
represents side view;
[0028] FIG. 2 contains schematic drawings of the counting grid
film, where FIG. 2(a) represents top view and FIG. 2(b) represents
side view;
[0029] FIG. 3 shows illustrations of various possible surface
features which form the grid lines, where FIG. 3(a) shows
triangular recess, FIG. 3(b) shows triangular top, FIG. 3(c) shows
recess with narrower bottom than its top, and FIG. 3(d) shows ridge
top with narrower top than its bottom;
[0030] FIG. 4 shows schematics of the spacer adhesive, where FIG.
4(a) is top view, FIG. 4(b) is side view of the spacer adhesive
with middle film, and FIG. 4(c) is side view of the spacer adhesive
without middle film; and
[0031] FIG. 5 depicts a typical grid line pattern that is similar
to the common improved Neubauer pattern.
[0032] These figures, which are idealized and are not to scale, are
intended to be merely illustrative and non-limiting.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Counting Chambers
[0034] FIG. 1 shows a counting device according to the present
invention. In this example, the counting device assembly has two
identical counting chambers. In general, any number of chambers can
be fabricated on a single assembly.
[0035] FIG. 1(a) depicts a complete counting device 10, with a top
part 20, a connecting layer 30, and a base part 40. On the top part
20, there is a sample introduction port 22 and an air escape port
24 for each counting chamber. FIG. 1(b) shows the interior of the
two counting chambers 26, and FIG. 1(c) is a top view. Typically,
the sample introduction port 22 and the air escape port 24 are
round holes in top part, and the diameter of the holes may be in
the range of 1 mm to 4 mm. The dimension of the chambers 26 may be
10 mm by 18 mm. The base part may be 25 mm by 75 mm, with a
thickness of 1 mm.
[0036] For each counting chamber, the area under which optical
observation must take place for counting cells are typically
halfway between the sample introduction port 22 and the air escape
port 24.
[0037] Top Part
[0038] The top part 20 is made of a film or sheet of plastic,
glass, or another rigid material. There are two ports 22 and 24 for
each counting chamber through the top part. The cell containing
solution is inoculated through one of the ports 22 while the
opposite port 24 allows the air to evacuate. The ports may be round
through holes in the top part, with a diameter of 2.4 mm. Suitable
diameters may be in the range of 1 mm to 4 mm. The thickness of the
top part 20 can be within the range of 0.01 mm to 3 mm, and is
preferably within 0.25 mm to 1 mm.
[0039] Connecting Layer
[0040] The connecting layer 30 may be made of a spacer film. As
shown in FIG. 1(d), the spacer film 30 consists three layers. The
middle layer 32 is a polymer film. Two layers 34 of pressure
sensitive adhesive (PSA) are coated on both the top and the bottom
side of the polymer film. The adhesive layers are protected by
polymer-coated paper-liner sheets, not shown here, one on either
side of the spacer film 30. The whole film stack is suitable for
die cutting. For the current invention, the spacer adhesive is cut
into shape, shown as 30 in FIG. 1(b) and as 60 in FIG. 4(a), using
a steel rule die.
[0041] During cell counting chamber assembling, the top PSA 34
adheres to the top part 20, while the bottom PSA 34 adheres to the
base part 40, and an enclosed chamber 26 is formed. The total
thickness of the PSA and polymer layers determines the chamber
height.
[0042] The total thickness of the spacer is chosen to be suitable
for cell counting. Different sized cells require different
thickness. It can range from 0.01 mm to 5 mm, preferably between
0.02 mm to 1 mm.
[0043] It is also possible to use only adhesives to define the
chamber height. This kind of adhesive comes with both protective
liners. The first liner is removed to allow one side of the
adhesive to adhere to the top part. The second liner is then
removed to join the top part to the base part.
[0044] Any kind of adhesive that can provide adequate adhesion
between the top and the base parts can be used. A preferable
adhesive system is the pressure sensitive adhesive, which comes as
a sheet format that can be cut into desired shapes.
[0045] FIG. 4 shows schematics of the spacer adhesive, where FIG.
4(a) is top view, FIG. 4(b) is side view of the spacer adhesive
with middle film, and FIG. 4(c) is side view of the spacer adhesive
without middle film.
[0046] One alternative to the spacer pressure sensitive adhesive is
to use adhesives dispersed with spacer beads. They are commonly
used in making liquid crystal displays by the display industry. In
this approach, beads with diameters equal to chamber gap are
dispersed into an adhesive pre-cursor solution. The mixture is then
applied onto defined locations on either the top part or the base
part to form the outlines of the counting chamber. The top part and
the base part are brought together and held in place when an energy
source (heat or radiation, such as ultraviolet light or e-beam)
solidifies the adhesive. The solidified adhesive bonds the two
parts together. The thickness of the adhesive, determined by the
spacer beads, defines the gap for the counting chamber.
[0047] Base Part
[0048] The base part 40 provides mechanical strength for the
counter as well as optical clarity for counting operation. The
counter has to be flat and rigid so that (1) the counting grid is
in focus under the field of view of an optical microscope, and (2)
the layer of cell-containing liquid is kept at a defined thickness.
The base 40 should not introduce any visual defect that interferes
with cell counting, both from its surface and inside its bulk. The
base part 40 must be optically clear so that light can pass through
the base to the cell-containing liquid inside the counting chamber
26.
[0049] Almost any material can be used for the base, as long as
that material is substantially optically clear, having good
strength and thermal stability. A microscope slide or any
transparent plastic sheet or glass can be used as the base.
Examples of suitable plastic materials include triacetate film,
vinyl film, and vinyl-PET laminates.
[0050] Counting Grid
[0051] The counting grid defines areas for cell counting. FIG. 5
shows one design of the counting grid that is suitable for cell
counting. The grid can be incorporated into either the top part 20
or the base part 40 of the counting chamber. Furthermore, for
focusing the gridlines and cells simultaneously within the depth of
field of the microscope, the counting grid is preferably
incorporated into the inside of the counting chamber. If the
counting grid is incorporated into the top part 20, the counting
grid is preferably incorporated into the bottom side of the top
part 20. Conversely, if the counting grid is incorporated into the
base part 40, the counting grid is preferably incorporated into the
top side of the base part 40.
[0052] For ease of manufacture, preferably the counting grid is
incorporated into a grid film. The grid film is a transparent film
with a grid pattern that defines the counting area. The most
economical material for the grid film is a plastic film. The grid
pattern may be formed by applying a coating of the uncured flowable
polymerizable solution to one surface of a base film, and then
contacting the uncured flowable polymerizable coating with a
configured molding surface having a series of cavities of the
desired pattern. Pressure is applied to cause the flowable uncured
polymerizable solution to fill the cavities of the molding surface,
and the solution is maintained in contact with the molding surface
while the coating is exposed to a curing agent to cause it to cure
and harden. The curing agent may be ultraviolet light, electron
beam, or heat. Once the coating has been sufficiently cured, it is
separated from the molding surface. The patterned polymer layer is
adherently and permanently bonded to the base film and the
composite sheet material forms the grid film.
[0053] Creation of microscopic features in plastic material from a
molding surface has been described in the prior art. Process of
making optical disks with microscopic features using
photo-polymerizable materials is disclosed in U.S. Pat. No.
4,374,077. Microstructure-bearing composite plastic articles and
method of making said articles are disclosed in U.S. Pat. No.
5,175,030. Grid lines directly molded onto the plastic for cell
counting is disclosed in U.S. Pat. No. 4,997,266. With the molding
method, the grid lines can only be built into a rigid part,
governed by the injection molding process. Another method called
hot stamping or embossing has been used to create holographic
films. In the embossing process, a plastic film is pressed against
a heated negative molding surface. The plastic in contact with the
tool is hot enough to flow, filling the cavities on the surface of
the tool. Upon cooling, the plastic film is removed from the tool,
bearing features that are negative topography of the mold. A
variation of the embossing method is to melt the plastic and cast
it onto a tool with a negative molding surface.
[0054] The tool that provides the negative molding surface can be
made from polymeric, metallic, composite, or ceramic materials. In
some instances, the polymerizable material may be cured by
radiation being applied through the tool. In such instances, the
tool should be sufficiently transparent to permit irradiation of
the polymerizable material. For features that are in the range of
smaller than 50 micrometer, photolithographic methods can be used
to define the features. With this method, a uniform layer of
photo-sensitive material is coated onto a surface. Certain areas of
the photo-sensitive material is exposed to light and later removed
from the surface. In some cases, the patterned photo-sensitive
material is adequate as a tool. In other cases, a chemical etching
process is applied to remove part of the underlying etchable
surface that is not protected by the photo-sensitive material.
After etching, the whole area is stripped of the photo-sensitive
material, exposing the surface pattern formed on the underlying
etchable material.
[0055] Illustrative examples of the photo-sensitive material are
photoresists commonly used in semiconductor processing. The
etchable material can be a metal, such as Al, Cu, Cr, or other
materials used for fabricating photomask, such as ion oxide,
aluminum oxide. The etchable material can also be inorganic sheets
or films, such as SiO.sub.2, Si.sub.3N.sub.4, TiO.sub.2. The
etchable material can also be glass.
[0056] FIG. 2 contains schematic drawings of the counting grid
film, where FIG. 2(a) represents top view and FIG. 2(b) represents
side view. FIG. 3 shows illustrations of various possible surface
features which form the grid lines, where FIG. 3(a) shows
triangular recess, FIG. 3(b) shows triangular top, FIG. 3(c) shows
recess with narrower bottom than its top, and FIG. 3(d) shows ridge
top with narrower top than its bottom.
[0057] Example of Making Cell Counting Devices
[0058] A. Counting Grid Film
[0059] The counting grid film consists of two layers. The base
substrate is a 10-mil (namely, 0.010 inch thick) polycarbonate
film. It was purchased from Tekra Co. The second layer is a clear
plastic with grid lines formed on its surface.
[0060] The process of fabricate the grid film consists of the
following steps:
[0061] (1) Make a master tool that has a negative image of the grid
line pattern.
[0062] (2) Prepare a UV polymerizable solution.
[0063] (3) Cast the UV polymerizable solution between the base
substrate and the master tool.
[0064] (4) Expose the assembly made in step (3) with UV light,
resulting in a film that has a grid pattern, as depicted in FIG.
5.
[0065] (5) Remove the grid film from the master tool.
[0066] Steps (3) to (5) can be repeated to produce multiple grid
films.
[0067] B. Top Film
[0068] The counting grid film is cut to size and placed within a
hole-punching die set. Two holes of 2.4 mm in diameter are cut
through the grid film.
[0069] C. Spacer Adhesive
[0070] The spacer adhesive is cut to dimension of 10 mm by 18 mm
using a steel rule die. The cutout area is then removed.
[0071] D. Microscope Slide is Used as the Base
[0072] E. Assemble the Cell Counter
[0073] The top film is laminated to one side of the spacer adhesive
after removing one protective liner from the adhesive. The combined
part is then laminated to glass slide after removing the protective
liner from the other side of the spacer adhesive.
1TABLE 1 Summary of components and raw materials Name Material
Product Information Vendor Spacer 3M Membrane 7945MP 3M Company
adhesive Switch Spacer St Paul, Minnesota Base substrate Bayer DE
1-1D Makrofo Tekra Co. Polycarbonate polycarbonate film New Berlin,
film Gloss/Gloss with liners WI UV Lens bond SK-9 Summers
polymerizable Optical solution Hatfield, PA Microscope Glass
(Commodity) (Commodity) slide
[0074] Example of Counting Cells
[0075] The concentration of Human T Lymphocyte cells were measured
using both the cell counter described in the present invention
(Model CG2 Cellometer.TM. from Nexcelom Bioscience LLC, Lawrence,
Mass.) and a common hemacytometer. The Model CG2 disposable cell
counter consists of a counting grid with sample introduction and
air escape ports, and a glass base.
[0076] Both CG2 disposable cell counter and the Hemacytometer have
counting grids depicted in FIG. 5. The liquid volume under each
corner square is 10.sup.-4 ml. The total number of cells in all
four corner squares are counted. The cell concentration is
calculated by the average number of cells in the corner square
divided by 10.sup.-4 ml.
[0077] The hemacytomer is cleaned after each use with alcohol.
Multiple CG2 counting chambers were used for counting and were
disposed after single use. The steps of this experiment are listed
as follows.
[0078] Step 1: Mix cell containing solution.
[0079] Step 2: Load cell counting chambers.
[0080] Step 3: Count the total number of cells inside the counting
grid.
[0081] Step 4: Dispose used CG2 cell counter or wash used
hemacytometer.
[0082] Step 5: Obtain cell concentration by dividing the total
number of cell counted by the volume of liquid.
[0083] Results:
2 Cellometer CG2 Hemacytomer Number of tests 17 17 Mean cell
concentration 1.87 .times. 10.sup.5 1.82 .times. 10.sup.5
(cells/ml)
[0084] In conclusion, the cell counting results using CG2, which is
made according to the present invention, and hemacytomer were
equivalent. With CG2, sample loading was simple and consistent.
There was no washing and drying involved in using CG2, which
eliminated the potential biohazard.
[0085] Various modifications and alterations of this invention will
become apparent to those skilled in the art without departing from
the scope and spirit of this invention.
* * * * *